Inducible Heterologous Expression Of Human Î±7-Nicotinic Acetylcholine Receptors In A Native Nicotinic Receptor-Null Human Clonal Line
Tetracycline-regulated expression of recombinant nicotinic acetylcholine receptors (nAChR) composed of human Î±7 subunits is achieved in native nAChR- null SH-EP1 human epithelial cells. Î±7 subunits are heterologously expressed as messenger RNA and as components of 125I-labeled Î±-bungarotoxin (I- Bgt)-binding nAChR (~ 10 pmol per milligram of membrane protein) at levels sensitive to the amount of tetracycline in cell growth medium. I-Bgt-binding Î±7-nAChR appear on the cell surface pool and in intracellular pools. The pharmacological profile for drug competition toward I-Bgt binding to these recombinant Î±7-nAChR matches that of human native Î±7-nAChR naturally expressed in SH-SY5Y human neuroblastoma cells (rank order potency methyllycaconitine > 1,1-dimethyl-4-phenylpiperazinium > (-)nicotine > cytisine > carbamylcholine â‰¥ D-tubocurarine). Chronic exposure to nicotine induces up-regulation of human recombinant Î±7-nAChR (80% up-regulation at 10 Î¼M nicotine) just as it does native Î±7-nAChR in other human cell lines. These studies confirm expression of nAChR as homooligomers of human Î±7 subunits from transgenes, establish a native nAChR-null background for such expression, and demonstrate that this expression can be regulated to facilitate studies of human Î±7-nAChR.
Digital Object Identifier (DOI)
Peng, Jian Hong; Lucero, Linda; Fryer, John; Herl, Jennifer; Leonard, Sherry S.; and Lukas, Ronald J., "Inducible Heterologous Expression Of Human Î±7-Nicotinic Acetylcholine Receptors In A Native Nicotinic Receptor-Null Human Clonal Line" (1999). Translational Neuroscience. 233.